The long-term objective of this project is to develop a comprehensive mechanistic understanding of intracellular transport processes that require the collective function of motor proteins. An optical trapping instrument will be employed to investigate the force production and nanometer-scale stepping mechanics of defined motor systems composed of exactly two kinesin motors. The use of the proposed motor systems alleviates several experimental issues that currently limit examination of collective motility by providing explicit knowledge of the composition and organization of motors on their cargo. Consequently, this work will allow essential collective transport parameters to be precisely measured, and complex behaviors of multi-motor systems to be reliably interpreted. In doing so, the proposed project will shed new light on mechanisms that participate in the regulation of cargo motion in cells. These aspects of collective motor transport will be investigated by accomplishing the following aims: Specific Aim 1: Determine the influence of the elasticity of motor-motor connections and inter-motor spacing on the collective force production by two interacting kinesin-1 molecules. Specific Aim 2: Resolve the molecular-scale mechanics of motor assemblies by monitoring collective stepping behaviors and multi-state, microtubule-attachment/detachment kinetics under constant optical and viscous loads. Public Health Relevance: Because motor proteins transport a diverse array of essential commodities to their required destination in cells, their malfunction can impair many vital cellular processes and, as a consequence, contribute to the pathology of several neurodegenerative diseases. The proposed study will identify and characterize key behaviors of motor proteins that must be known to illuminate how compromised motor transport can lead to diseased cellular states.